purpose. To determine whether the subretinal space can extend immune privilege
to allogeneic tumor cell grafts that do not possess their own inherent
immune privilege.

methods. P815 tumor cells were injected into the anterior chamber (AC), the
subretinal (SR) space, or subconjunctivally in eyes of BALB/c
(allogeneic), SCID (immune incompetent), normal DBA/2
(syngeneic), or DBA/2 mice presensitized with P815 cells transfected
with interleukin-12 and B7.1. Tumor growth was observed clinically and
histologically for up to 50 days. BALB/c recipients were tested for
suppression of DBA/2-specific delayed hypersensitivity and concomitant
immunity. The SR space of tumor-containing eyes was assessed for its
capacity to support ovalbumin (OVA)-specific anterior chamber
associated immune deviation (ACAID).

results. P815 cells injected into the SR space of presensitized and normal DBA/2
and SCID mice grew progressively, resulting eventually in recipient
death. Tumor cells injected into the SR space of eyes of BALB/c mice
grew progressively until day 14, followed by tumor regression resulting
in phthisis bulbi (14/35) or tumor elimination (19/35)
with preserved ocular anatomy by day 35. Despite elimination of tumors
from the SR space, BALB/c recipients exhibited DBA/2-specific ACAID and
concomitant immunity. In addition, OVA injected into the SR space of
eyes from which tumor has been eliminated induced ACAID.

conclusions. Various parameters of immune privilege, originally described for the
AC, are characteristic of immune privilege within the SR space.
However, because P815 cells placed in the AC prove lethal for BALB/c
recipients, but P815 cells placed in the SR space resolve without
jeopardizing the host’s life, immune privilege in the SR space can be
distinguished from immune privilege in the AC, and this may have
implications for grafts of retinal tissue placed within the SR
space.

The anterior chamber (AC) of the murine eye has been studied
extensively for its property as an immune privileged
site.1234567 The experiments of Niederkorn et
al.6 first documented that minor histoincompatible tumor
cells injected into the AC formed progressively growing tumors that
never experienced immune rejection. Moreover, these allogeneic tumors
induced a deviant systemic immune response, termed anterior chamber
associated immune deviation (ACAID), which has proved to be a
characteristic feature of systemic immunity induced by a wide variety
of antigens injected intracamerally.24 Because
unrelenting tumor growth coincided temporally with sustained ACAID in
recipients of AC tumor injections, the concept emerged that deviant
immunity and immune privilege are mechanistically linked. This concept
was further strengthened by the observations that major
histocompatibility complex (MHC) incompatible tumor cell grafts
injected into the AC experienced immune privilege only
transiently,8 and, correspondingly, the ACAID induced by
MHC incompatible tumors also proved to be evanescent.8 Immune privilege is also a feature of the vitreous
cavity.9 The initial pattern of growth of P815 cells
injected in this compartment of eyes of BALB/c mice has been found to
be essentially similar to that observed for P815 tumors placed within
the AC, and P815 tumors growing within the vitreous cavity readily
induced ACAID.

The subretinal (SR) space is a third intraocular compartment with the
potential to be an immune privileged site.101112 Our
laboratory has had a long-standing interest in promoting the survival
of retina-derived grafts in the SR space; to aid in this goal we have
begun to examine the extent to which this intraocular compartment
displays immune privilege. Several years ago Jiang et
al.1113 reported that allografts of retinal pigment
epithelium (RPE) as well as of neonatal neuronal retina experienced
immune privilege within the SR space. Specifically, both RPE and
neonatal neuronal retina allografts survived for prolonged intervals
when injected into the SR space, compared with the subconjunctival
space. However, both RPE and neuronal retina grafts may themselves be
immune privileged tissues and, thus, capable on their own of resisting
immune rejection. Therefore, we cannot be sure that their prolonged
acceptance within the SR space is a property of the space or of the
grafts themselves. To assess directly the immune privileged status of
the SR space as a site, it is necessary to select allografts for SR
space implantation that do not display the inherent property of immune
privilege. P815 tumor cells represent such a graft.

Recently, we have reported that a single injection of P815 cells into
the SR space of eyes of BALB/c mice induces donor-specific
ACAID.12 Because ACAID induction is only one manifestation
of immune privilege, we elected to describe the growth pattern of P815
tumors implanted in the SR space of eyes of allogeneic recipients
(BALB/c), syngeneic recipients (DBA/2), and immune incompetent
recipients (SCID mice) as independent means to assess immune privilege
within the SR space. Our results revealed that tumors grew
progressively in the SR space of eyes of all recipients, confirming
that the SR space is an immune privileged site. However, in allogeneic
recipients, intraocular tumor masses began to resolve between 2 and 3
weeks post–tumor implantation, and, eventually, almost all mice were
cured of their tumors. Surprisingly, P815-specific ACAID persisted even
when tumors were eliminated. Additionally, the SR space of eyes that
recovered from tumor growth regained the capacity to support ACAID to
an unrelated antigen, implying that immune privilege persisted at the
site. The possible mechanisms that might be responsible for graft
rejection in a site that retains its immune privilege are discussed.

Materials and Methods

Animals

Adult female BALB/c, DBA/2, and CB.17 (SCID) mice, 6 to 8 weeks of
age, were obtained from the animal facilities at the Schepens Eye
Research Institute or from Jackson Laboratories (Bar Harbor, ME). Mice
were maintained in a common room of a vivarium. Inoculations,
injections, clinical examinations, and enucleations were conducted
under anesthesia induced by intraperitoneal injection of ketamine
(Ketalar; Parke–Davis, Paramus, NJ), 0.075 mg/g body weight, and
xylazine (Rompun; Pheonix Pharmaceutical, St. Joseph, MO), 0.006 mg/g
body weight. All experimental procedures conformed to the ARVO
Statement on the Use of Animals in Ophthalmic and Vision Research. Five
mice were used for each experimental group, and experiments were
repeated at least twice.

Intraocular Injections

P815 mastocytoma cells (DBA/2 origin) were grown as previously
described.6 For experiments, 2 ×
105 P815 cells were slowly injected into
different ocular sites in a volume of 2 μl using a glass micropipette
connected to a 10-μl syringe. For AC, and subconjunctival injections,
a 0.3-mm penetrating wound was made in the peripheral portion of the
cornea, 1 mm posterior to the limbus, or in the fornix of the
conjunctiva, respectively, using a 30-gauge needle. For injections into
the SR space, the temporal conjunctiva was opened parallel to the
limbus and the eye was rotated to expose the posterior part of the
sclera. A 0.3-mm tangential sclerotomy was made with a 30-gauge needle,
and a retinal bleb was created with 0.5 μl Hanks’ balanced salt
solution (HBSS), and then tumor cells or antigen-containing solutions
were injected through the wound into the SR space.

Soluble Antigen

Fifty micrograms of ovalbumin (OVA; Sigma Chemicals, St. Louis,
MO) in HBSS was used as soluble antigen and was injected in a volume of
2 μl into the eyes of experimental animals.

Clinical Evaluation of Tumor Growth of P815 Cells

The anterior segment of eyes after tumor cell inoculation was
examined clinically with a Topcon slit-lamp (Tokyo Optical, Tokyo,
Japan), and the posterior segment was examined through a coverslip with
a dissecting microscope. All eyes were examined every second day after
injection of tumor cells. Subretinal tumor growth was graded using the
following categories: 0, no tumor detectable; 1, minimal tumor (less
than 2 quadrants); 2, medium tumor (less than 3 quadrants); 3, SR tumor
in more than 3 quadrants; 4, SR space filled with tumor and
infiltration of vitreous body; 5, eye filled with tumor (extension into
AC); 6, same as 5 plus protrusio bulbi and extraocular tumor
extension; and 7, death of animal with large orbital tumor.

Histopathologic Evaluation of Tumor Growth of P815 Cells

For histologic evaluation, tumor-bearing and control eyes of each
group were enucleated at different time points (3, 7, and 14, 21, 45
days), fixed with 10% buffered formalin, processed for routine
histopathology, and stained with hematoxylin and eosin.

Sensitization of DBA/2 Mice to P815 Tumor Antigens

Immunization of mice with P815 cells transfected with B7.1 and
interleukin (IL)-12 allows recipients to reject wild-type P815 cells
injected into a conventional site.14 For this purpose P815
tumor cells had been previously transfected with vectors containing
genes encoding the co-stimulatory molecules B7.1 and
IL-12.14 The transfected cells were used to sensitize
groups of DBA/2 mice to the tumor-specific antigens of P815 before
intraocular implantation of P815 cells. For sensitization, transfected
P815 cells (1 × 106) were injected into the
flank of normal DBA/2 mice 14 days before experimental study.

Assay for Delayed Hypersensitivity

Mice to be assayed for P815-specific delayed hypersensitivity (DH)
received intracameral, subretinal, or subconjunctival injections of
P815 cells at various times before assay. At day 45 after initial
injection of P815 cells, 5 × 105 x-irradiated (2000 R) P815 tumor cells in 10 μl were injected into
the left ear pinnae of mice with intact ocular morphology. For mice to
be assayed for OVA-specific DH, OVA (100 μg) combined with complete
Freund’s adjuvant (CFA) was injected subcutaneously within 7 days of
intraocular injection of OVA. Ten days later, 200 μg OVA in 10 μl
was injected into the left ear pinnae of the mice. The right ear served
as untreated control. Both ear pinnae were measured immediately before
injection and 24 hours later with an engineer’s micrometer (Mitutoyo,
Tokyo, Japan). The measurements were performed as triplicates. Results
were expressed as specific ear swelling = (24-hour
measurement − 0-hour measurement) experimental ear − (24
hour measurement − 0-hour measurement) negative control ear × 10–3 mm. A two-tailed Student’s t-test was used and significance assumed if P < 0.05.

Results

Pattern of Growth and Survival of P815 Cells in SR Space of BALB/c
Eyes

Panels of normal BALB/c mice received injections of P815 cells (2×
105/site) into the SR space, the AC, or
subconjunctivally. The growth of these tumors was examined by direct
inspection, by slit-lamp, by indirect ophthalmoscopy, and, eventually,
by histology of enucleated tumor-bearing eyes. As described
previously,12 tumors grew progressively within the AC and
the SR space (Fig. 1A , Table 1 ) through day 14. By day 14 post-implantation, tumor
cells had broken through the sclera and penetrated into the orbit (Fig. 1B) . In contrast, P815 cells injected subconjunctivally formed
transiently growing masses that promptly receded and were no longer
evident at 14 days (Table 1) . Beyond 14 days, the patterns of tumor
growth in the AC and SR space began to differ. Whereas the AC tumors
simply continued to grow progressively beyond day 21 post-implantation,
SR space tumors began to recede when examined at this time point (Table 1) . Eyes with P815 cells in the SR space then progressed toward one of
two outcomes: phthisis bulbi was observed by day 30 in 19 of
35 eyes; complete tumor elimination with preserved ocular morphology
was observed in 14 of 35 eyes (Table 1) . Two eyes showed slowly
progressing tumor growth that was still evident when the experiment was
terminated on day 60 post-implantation. These findings indicate that
immune privilege was extended equally well to allogeneic tumor cells
injected into the AC and SR space for the first 2 weeks after
implantation. Thereafter, privilege was apparently lost in the SR
space, and as a consequence the tumors (with or without the eye) were
destroyed.

Pattern of Growth and Survival of P815 Cells in SR Space of Eyes of
Syngeneic and Immune Incompetent Mice

One explanation for the different patterns of survival of P815
cells in the SR space and AC of eyes of BALB/c mice may be that
unrelenting tumor cell growth is not possible in the SR space because
of space and nutritional constraints, rather than because of loss of
immune privilege. To examine this possibility, we injected 2 ×
105 P815 cells into the SR space of eyes of CB.17
(SCID) mice, which are profoundly immune incompetent, and in the eyes
of DBA/2 mice, which are syngeneic with the tumor and therefore only
exhibit weak immunity that is directed against tumor-specific antigens.
For comparison’s sake, P815 cells were also implanted in the AC of a
separate panel of normal DBA/2 mice. As revealed in Figure 2 , progressively growing tumors formed in the SR space of both groups of
recipients, and all mice eventually died of the tumor. In SCID mice,
tumor growth was extremely rapid and mice died between 8 and 12 days
post-implantation, presumably due to systemic dissemination of the
tumor. In DBA/2 mice, the rate of tumor growth in the AC and the SR
space was similar, although tumor growth in the SR space tended to lag
behind. Eventually, however, the same outcome was reached. Their
intraocular tumors penetrated through the ocular capsule, forming large
orbital tumors. These mice died of direct extension of tumor cells into
the brain, a mode of death similar to that of mice with P815 cells in
the AC. The findings that SCID mice died from SR space P815 cells
within 12 days, whereas DBA/2 mice survived significantly longer,
indicate that adaptive immunity acts to suppress intraocular tumor
growth, even though that immunity may be weak and directed at
tumor-specific antigens. Progressive tumor growth in the eyes of DBA/2
mice without evidence of elimination argues against the existence of
either a microanatomic or a nutritional barrier to progressive tumor
growth in the SR space. Thus, the elimination of tumors from the SR
space of BALB/c eyes must reflect allograft rejection and, presumably,
the loss of immune privilege at the site.

Pattern of Growth and Survival of P815 Cells in SR Space of Eyes of
Presensitized Syngeneic Mice

Because P815 cells injected into the flank of DBA/2 mice (a
non–immune privileged site) form progressively growing tumors,
progressive growth after injection into the AC or SR space may not be a
reflection of immune privilege. Recently, Chen et al.14 have reported that DBA/2 mice can be sensitized to tumor antigens by
receiving a subcutaneous injection of P815 cells that had been
transfected with B7.1 and IL-12. DBA/2 mice primed with B7.1 and
IL-12–transfected P815 cells acquire the ability to reject wild-type
P815 cells injected into the flank.14 However, Chen et al.
also reported that when wild-type P815 cells were injected into the AC
of similarly primed DBA/2 mice, the tumors grew progressively,
indicating that immune privilege is extended to weakly antigenic tumor
placed in this intraocular site, even in the presence of preexistent
immunity. We next examined whether the SR space more closely resembled
a privileged site (AC) or a nonprivileged site (subconjunctival space)
by using as recipients DBA/2 mice that had been immunized with B7.1 and
IL-12–transfected P815 cells. In contrast to cells injected into the
subconjunctival space, P815 cells were able to create progressively
growing tumors in the SR space of presensitized DBA/2 recipients, and
the majority of the mice (8/10) eventually died (Fig. 2) . This result
confirms that the normal SR space shares with the AC the capacity to
extend immune privilege to weakly antigenic tumors even when recipients
have been specifically presensitized to tumor-specific antigens. The
growth of P815 tumor cells in the AC or the SR space tended to be
slower in presensitized DBA/2 mice when compared with naïve
DBA/2 mice (Fig. 2) , indicating that persisting immunity directed
against tumor antigens has a measurable effect on tumor growth in the
eye. Eventually, however, this immunity fails and immune privilege is
able to promote unlimited tumor growth.

Systemic DH Response to P815 Cells in SR Space of Eyes of BALB/c
Mice

We have recently reported that P815 cells placed in the SR space
of eyes of BALB/c mice induce donor-specific ACAID that is detectable
within 14 days.12 We wished to determine whether
suppressed donor-specific DH (i.e., ACAID) persisted in mice whose
intraocular tumors had undergone regression. To examine this point,
panels of BALB/c mice received P815 cells in the SR space or
subconjunctivally (positive controls). After 14, 22, or 47 days, panels
of these mice received an intrapinnae injection of x-irradiated P815
cells (5 × 105). Ear swelling responses as
evidence of DH were evaluated 24 and 48 hours later. As the results
displayed in Figure 3 reveal, impaired DH was observed among recipients of P815 cells in the
SR space, whether the mice were tested at 14, 22, or 47 days. As
before, tumors were eliminated in virtually all these eyes by 30 days,
indicating that loss of intraocular tumor did not correlate with
acquisition of donor-specific DH. Moreover, DH was equally impaired in
mice with phthisis bulbi and mice with anatomically
preserved eyes from which tumor had been eliminated. The finding that
donor-specific DH did not emerge in mice that eliminated their
intraocular tumors from the SR space means that tumor rejection was not
procured by T cells that mediate donor-specific DH.

Capacity of SR Space of Eyes with Resolved Tumors to Support ACAID
Induction

The results presented above, documenting persistence of
donor-specific ACAID in mice whose intraocular tumors had disappeared,
do not directly demonstrate that these eyes had recovered the property
of immune privilege. To assess whether SR spaces from which P815 tumors
had been eliminated still possessed the property of immune privilege,
we tested whether an irrelevant antigen could evoke a deviant systemic
immune response if injected into the SR space of eyes from which tumors
had spontaneously resolved, and which displayed intact ocular
morphology. Eyes that had undergone phthisis bulbi after
tumor elimination were not used in these experiments. Ovalbumin (50μ
g) was injected into the SR space of recovered eyes at day 45 after
P815 cell injection. By that time, SR tumors had been completely
eliminated, and SR space had recovered with scar formation in some
mice. Seven days later, these mice received a subcutaneous immunization
with OVA and CFA. Ten days later, the ear pinnae of these mice were
challenged with OVA (200 μg), and ear swelling was evaluated 24 and
48 hours thereafter. The results of a representative experiment (of
three) are presented in Figure 4 . Ovalbumin-specific DH was not detected in mice that received OVA into
recovered SR spaces. By contrast, mice immunized with OVA/CFA without
prior intraocular injection of OVA displayed intense DH. Thus, the SR
space of eyes from which tumor had been eliminated displayed, or
perhaps regained, the capacity to support ACAID induction.

Capacity of P815 Cells in SR Space to Induce Concomitant Immunity

BALB/c mice that contain progressively growing P815 tumors
in one eye acquire concomitant immunity. That is, subsequent injection
of P815 cells into the flank, or even into the contralateral eye, fails
to result in tumor formation.15 Although the precise
mediators of concomitant immunity have not been elucidated, DH does not
contribute to protection against tumors. Concomitant immunity is not
only able to prevent the establishment of new tumors, but it appears to
be responsible for the inability of the original tumor in the eye to
seed systemic metastases.16 We next wished to determine
whether P815 cells injected into the SR space induced concomitant
immunity similar to that induced by P815 cells injected into the AC of
the eye. Accordingly, panels of BALB/c mice received P815 cells into
the SR space. Seven days later, a second inoculum of P815 cells (2 × 105) was placed into the AC of the right eye.
The pattern of tumor growth within that eye was observed and recorded
daily. Within a few days of the AC tumor cell injection, a haze
appeared in the AC, but no tumor mass was detected. Thereafter, the
haze in the AC cleared, but no visible tumor ever emerged (data not
shown). This result indicates that P815 cells placed in the SR space
induce concomitant immunity similar to tumors originally placed in the
AC.

Discussion

Our results provide several new pieces of evidence to support the
view that the SR space is an immune privileged site. First, P815 tumor
cells, which possess no inherent immune privilege, formed progressively
growing tumors when placed in the SR space of eyes of allogeneic BALB/c
mice. Second, P815 cells also formed progressively growing tumors when
placed in the SR space of eyes of syngeneic DBA/2 mice presensitized to
the weak tumor-specific antigens of this tumor cell line. Third, mice
bearing P815 tumors in the SR space displayed concomitant immunity
(i.e., they resisted tumor formation when P815 cells were injected into
the AC of the uninjected fellow eye). Fourth, P815 cells in the SR
space of BALB/c mice induced persistent DBA/2-specific ACAID, a
confirmation of our recent reported finding. We conclude that the SR
space is indeed an immune privileged site.

However, the tumors that formed intraocularly after SR space
injection of P815 cells into BALB/c mice did not persist and grow
indefinitely. Instead, the tumors, which had filled the globe and
infiltrated through the choroid and sclera, began to recede 14 to 21
days after implantation. In some cases, the tumors simply disappeared,
leaving the eye anatomically intact. In the other cases,
tumor-containing eyes underwent phthisis. In fact, no mice died from
tumors that were implanted in the SR space. This outcome contrasts
sharply with that of BALB/c mice in which P815 tumor cells were
implanted in the AC; all these mice die eventually of cerebral
extensions of the tumors. We considered two possible explanations for
the distinctive pattern of tumor growth and elimination in the SR space
of eyes of BALB/c mice. First, the SR space is incapable of supporting
sustained tumor growth. Second, immune privilege in the SR space may
not be permanent, as it usually is in the AC.

We doubt that tumors are eliminated from the SR space because this site
cannot sustain progressive tumor growth. This doubt is based on our
finding that P815 cells readily formed tumors that continued to grow in
the SR space of eyes of SCID mice and DBA/2 mice until their recipients
died. In DBA/2 mice, the tempo of tumor growth in the SR space was
virtually indistinguishable from that in the AC. Thus, the SR space is
capable of supporting tumor growth that is progressive and eventually
fatal. It is of interest that P815 cells were far more aggressive in
SCID mice than in DBA/2 mice. Although SCID mice are defenseless
against this tumor, T cells of DBA/2 mice are able to recognize weak
tumor-specific antigens on P815 cells. We presume that the extended
survival of DBA/2 mice bearing intraocular tumors, compared with SCID
mice, reflects the expression of this antitumor immune response.
However, this level of tumor immunity, which retarded intraocular tumor
growth, failed ultimately to protect against the tumor’s lethality.
The fact that tumor growth within DBA/2 eyes was less aggressive than
in SCID eyes indicates that tumor immunity must be able to express
itself in the SR space, even though this site is immune privileged.

The ability of immunity to express itself in the SR space supports the
second possible explanation for the elimination of tumor cells from
eyes of BALB/c mice (i.e., that immune privilege for P815 cells in the
SR space is transient). However, several other experimental
observations cast doubt on this explanation. First, injection of OVA
into the SR space of BALB/c eyes from which tumors had spontaneously
resolved induced ACAID. The recent literature indicates that the
existence of immune privilege in the AC is strongly correlated with the
capacity of the AC to support ACAID induction.17 Assuming
that a similar correlation exists for the SR space, the ability of the
recovered SR space to support OVA-specific ACAID implies that immune
privilege is also present. Second, BALB/c mice from whose eyes SR
tumors spontaneously resolved failed to acquire DBA/2-specific DH. In
previous reports, elimination of P815 tumors from eyes of mice
correlated with acquisition of donor-specific DH. In particular, P815
cells established tumors in the AC of MHC-incompatible C57BL/6 and A/J
mice; and during the first 14 days while the tumors were growing
progressively, the recipients displayed DBA/2-specific ACAID. However,
when the ocular tumors were rejected during the subsequent 7 to 10
days, the recipients suddenly acquired DBA/2-specific DH.8 Moreover, circumstantial evidence implicates T cells that mediate DH in
ocular tumor rejection.18 Thus, when grafts within immune
privileged sites are eliminated because of loss of privilege, the
systemic immune response shifts from a deviant one to a conventional
one. No such shift was detected when P815 tumors were eliminated from
the SR space of BALB/c eyes. For these reasons, we cannot be sure that
the reason for tumor elimination from the SR space of BALB/c eyes is a
change in the immune privileged status of the site.

If the SR space is physically capable of supporting progressive
unrelenting P815 tumor cell growth (as our evidence revealed in
syngeneic DBA/2 mice), and if resolution of the tumor in eyes of BALB/c
mice does not result from loss of immune privilege in the SR space, how
then can the elimination of these intraocular tumors be explained?
Unique anatomic features of the SR space might contribute to the tumor
elimination, which is not seen after AC injection of tumor cells.
Moreover, the trauma necessary to insert cells into the SR space is
considerably greater than that within the AC. In the former, the
choroid must be penetrated, and the retinal detachment created by the
procedure may further alter the properties of the space. However, even
after this surgical trauma, the SR space displays the capacity to
support the induction of immune deviation and to grant immune
privilege, at least for a temporary period. The difference in tumor
growth begins to be apparent when the tumor penetrates and destroys the
normal anatomy of the original injection site. Tumors originally placed
in the AC break out and continue their unrelenting growth. However,
when SR tumors break out of the space, further growth stops and the
tumor begins to recede.

A third possibility is suggested by recent experiments reported
by Ksander et al.19 These investigators have discovered
that P815 cells growing in the immune privileged AC acquire (through
time) unique properties that enable the eye-derived tumor cells to grow
progressively when implanted anywhere, even at nonprivileged sites. The
evidence suggests that tumor cells growing within the AC may assume the
status of an “immune privileged tissue.” If this interpretation of
the data of Ksander et al. is correct, then we must consider the
possibility that mechanisms responsible for immune privilege in the AC
and SR space are similar but not identical. Particularly, the SR space
may differ from the AC by not possessing the ability to confer“
immune privilege” on tumor cells placed within. Experiments are
currently under way to test the hypothesis that immune privilege in the
SR space lacks the capacity to confer “immune privilege” on P815
cells placed within this site.

Whatever the mechanism, our evidence indicates that immune
privilege is first extended, then withdrawn, from allogeneic tumor
grafts placed in the SR space. This information is likely to be
relevant to the fate of allogeneic retinal tissues grafted into the SR
space. As Jiang et al. reported previously,11 allogeneic
neonatal retina grafts experience immune privilege when implanted in
the SR space of eyes of normal mice. The recipients even develop
donor-specific ACAID within 2 weeks of implantation. However, these
neuronal retina allografts eventually fail in the AC as well as in the
SR space, and their failure is somehow linked to the development of
destructive anti-donor immunity.20 The results of Jiang et
al. strongly resemble our experience with allogeneic tumor grafts in
the SR space in that both types of grafts are ultimately destroyed
(i.e., immune privilege is withdrawn). Working out the pathogenesis of
this withdrawal may provide insights into how to extend indefinitely
the survival of allogeneic retinal tissue placed in the SR space.

Supported by Deutsche Forschungsgemeinschaft Grant WE 1462/3-1/2; by
United States Public Health Service Grant EY 09595; and by the Minda
DeGunzburg Research Center for Retinal Transplantation.

Submitted for publication December 29, 1998; revised April 16 and July 12, 1999; accepted July 14, 1999.

Observation of tumor growth after injection of P815 cells into
the SR space, the AC, or subconjunctivally in BALB/c mice. Tumor growth
was evaluated at days 14, 21, and 30 after P815 cell injection. Tu.,
tumor.

P815 specific DH measurement at different time points after injection
of P815 cells into the SR space of BALB/c mice. Ear swelling analysis
24 hours after ear challenge with P815 cells in animals with
inoculation of P815 cells into the SR space. P815 inoculation had been
performed 14 days SR(14d), 22 days SR(22d), or 47 days SR(47d) before
measurement. As positive control, animals received P815 cells
subconjunctivally (Scon). Naive animals with ear challenge only served
as negative control (negative). * indicates mean response significantly
lower than positive control, P < 0.05.

Figure 3.

P815 specific DH measurement at different time points after injection
of P815 cells into the SR space of BALB/c mice. Ear swelling analysis
24 hours after ear challenge with P815 cells in animals with
inoculation of P815 cells into the SR space. P815 inoculation had been
performed 14 days SR(14d), 22 days SR(22d), or 47 days SR(47d) before
measurement. As positive control, animals received P815 cells
subconjunctivally (Scon). Naive animals with ear challenge only served
as negative control (negative). * indicates mean response significantly
lower than positive control, P < 0.05.

Ovalbumin-specific DH measurement after OVA injection into the SR space
after elimination of P815 cell tumors. Ear swelling analysis was
performed 24 hours after ear challenge with OVA in animals with
subcutaneous immunization with OVA and CFA 7 days earlier. One week
before immunization, OVA had been injected into the SR space
SR(naïve). One group of animals had received an injection of
P815 cells in the SR space and had rejected the SR tumor before OVA
injection SR(tumor). Negative control animals (negative) received no
immunization. Positive control mice were only immunized (positive). *
indicates mean response significantly lower than positive control, P < 0.05.

Figure 4.

Ovalbumin-specific DH measurement after OVA injection into the SR space
after elimination of P815 cell tumors. Ear swelling analysis was
performed 24 hours after ear challenge with OVA in animals with
subcutaneous immunization with OVA and CFA 7 days earlier. One week
before immunization, OVA had been injected into the SR space
SR(naïve). One group of animals had received an injection of
P815 cells in the SR space and had rejected the SR tumor before OVA
injection SR(tumor). Negative control animals (negative) received no
immunization. Positive control mice were only immunized (positive). *
indicates mean response significantly lower than positive control, P < 0.05.

P815 specific DH measurement at different time points after injection
of P815 cells into the SR space of BALB/c mice. Ear swelling analysis
24 hours after ear challenge with P815 cells in animals with
inoculation of P815 cells into the SR space. P815 inoculation had been
performed 14 days SR(14d), 22 days SR(22d), or 47 days SR(47d) before
measurement. As positive control, animals received P815 cells
subconjunctivally (Scon). Naive animals with ear challenge only served
as negative control (negative). * indicates mean response significantly
lower than positive control, P < 0.05.

Figure 3.

P815 specific DH measurement at different time points after injection
of P815 cells into the SR space of BALB/c mice. Ear swelling analysis
24 hours after ear challenge with P815 cells in animals with
inoculation of P815 cells into the SR space. P815 inoculation had been
performed 14 days SR(14d), 22 days SR(22d), or 47 days SR(47d) before
measurement. As positive control, animals received P815 cells
subconjunctivally (Scon). Naive animals with ear challenge only served
as negative control (negative). * indicates mean response significantly
lower than positive control, P < 0.05.

Ovalbumin-specific DH measurement after OVA injection into the SR space
after elimination of P815 cell tumors. Ear swelling analysis was
performed 24 hours after ear challenge with OVA in animals with
subcutaneous immunization with OVA and CFA 7 days earlier. One week
before immunization, OVA had been injected into the SR space
SR(naïve). One group of animals had received an injection of
P815 cells in the SR space and had rejected the SR tumor before OVA
injection SR(tumor). Negative control animals (negative) received no
immunization. Positive control mice were only immunized (positive). *
indicates mean response significantly lower than positive control, P < 0.05.

Figure 4.

Ovalbumin-specific DH measurement after OVA injection into the SR space
after elimination of P815 cell tumors. Ear swelling analysis was
performed 24 hours after ear challenge with OVA in animals with
subcutaneous immunization with OVA and CFA 7 days earlier. One week
before immunization, OVA had been injected into the SR space
SR(naïve). One group of animals had received an injection of
P815 cells in the SR space and had rejected the SR tumor before OVA
injection SR(tumor). Negative control animals (negative) received no
immunization. Positive control mice were only immunized (positive). *
indicates mean response significantly lower than positive control, P < 0.05.

Observation of tumor growth after injection of P815 cells into
the SR space, the AC, or subconjunctivally in BALB/c mice. Tumor growth
was evaluated at days 14, 21, and 30 after P815 cell injection. Tu.,
tumor.

Copyright 1999 The Association for Research in Vision and Ophthalmology, Inc.